Mauro Jesus Bronzatto

Adenosine receptors co-operate with NMDA preconditioning to protect cerebellar granule cells against glutamate neurotoxicity.

N-Methyl-D-aspartate (NMDA) preconditioning is evoked by subtoxic concentrations of NMDA (50 microM), which has been shown previously to lead to transient resistance to subsequent lethal dose of glutamate or NMDA in cultured neurons. The purpose of this study was to investigate the participation of adenosine A1 and A2A receptors on NMDA preconditioning against glutamate-induced cellular damage in cerebellar granule cells. NMDA preconditioning prevented the stimulatory effect induced by glutamate on AMP hydrolysis, but not on ADP hydrolysis. The neuroprotection evoked by NMDA preconditioning against glutamate-induced cellular damage was prevented by the presence of adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT, 100 nM), but not by the adenosine A2A receptors antagonist, (4-(2[7-amino-2-(2-furyl {1,2,4}-triazolo{2,3-a{1,3,5}triazian-5-yl-aminoethyl)phenol (ZM 241385, 50 nM). Interestingly, a long-term treatment with CPT or ZM 241385 alone protected cells against glutamate-induced neurotoxicity. Moreover, the functionality of adenosine A1 receptor was not affected by NMDA preconditioning, but this treatment promoted adenosine A2A receptor desensitization, measured by cAMP accumulation. Taken together, the results described herein suggest that the neuroprotection evoked by NMDA preconditioning against cellular damage elicited by glutamate occurs through mechanisms involving adenosine A2A receptors desensitization co-operating with adenosine A1 receptors activation in cerebellar granule cells.

The modulation of ecto-nucleotidase activities by glutamate in cultured cerebellar granule cells

Several enzymes hydrolyze ATP, producing ADP which is hydrolyzed to AMP. Ecto-5′-nucleotidase produces adenosine from AMP. Glutamate (Glu) is an excitatory neurotransmitter and increases extracellular adenosine levels, which is considered an important inhibitory neuromodulator. Here we show that Glu activates ADP and AMP hydrolysis. NMDA and kainic acid (KA) also increased these enzymatic activities, but 1-aminocyclopentane-1S, 3R-dicarboxylic acid (ACPD) had no effect. Dihydrokainate (DHK), an inhibitor of glutamate uptake, also blocked glutamate-evoked activation of ecto-nucleotidases, suggesting that this activation was also Glu transporters dependent. Therefore, we suggest that the Glu-evoked stimulation of ecto-nucleotidases might contribute to the increase of adenosine in extracellular space induced by Glu.

Effect of the L- or D-aspartate on ecto-5′nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A2A receptors

Summary.Glutamate increases the extracellular adenosine levels, an important endogenous neuromodulator. The neurotoxicity induced by glutamate increases the ecto-5′-nucleotidase activity in neurons, which produces adenosine from AMP. L- and D-aspartate (Asp) mimic most of the actions of glutamate in the N-methyl-D-aspartate (NMDA) receptors. In the present study, both amino acids stimulated the ecto-5′-nucleotidase activity in cerebellar granule cells. MK-801 and AP-5 prevented the L- and D-Asp-evoked activation of ecto-5′-nucleotidase. Both NMDA receptor antagonists prevented completely the damage induced by L-Asp, but partially the D-Asp-induced damage. The antagonist of adenosine A2A receptors (ZM 241385) prevented totally the L- Asp-induced cellular death, but partially the neurotoxicity induced by D-Asp and the antagonist of adenosine A1 receptors (CPT) had no effect. The results indicated a different involvement of NMDA receptors on the L- or D-Asp-evoked activation of ecto-5′-nucleotidase and on cellular damage. The adenosine formed from ecto-5′-nucleotidase stimulation preferentially acted on adenosine A2A receptor which is probably co-operating with the neurotoxicity induced by amino acids.

Effects of glutamate transporter and receptor ligands on neuronal glutamate uptake

The excitatory amino acids (EAAs) transporters regulate the balance between physiological and pathological signaling over stimulation of the glutamatergic system pathway. The effect of transportable substrates and glutamate (Glu) receptor agonists on Glu uptake in neuronal cells was assessed at different conditions. Cells pre-incubated with Glu, L- or D-aspartate (Asp) and washed presented an inhibition on [(3)H]-Glu uptake and this effect was not mimicked by Glu receptors agonists. The effects of L- and D-Asp were not altered by the presence of N-methyl-d-aspartate (NMDA) receptor antagonists. Thus, the reduction on Glu uptake induced by EAAs is probably linked to the transporter activity. In contrast, the presence of NMDA or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) during the pre-incubation and the [(3)H]-Glu uptake assay period increased Glu uptake, whilst kainic acid (KA) had no effect. The NMDA effect was not altered by its antagonists (+/-)-2-amino-5-phosphonopentanoic acid (AP-5) or dizocilpine (MK-801). The SR-ACPD effect was due to the activation of metabotropic Glu receptor, since it was abolished by its antagonist, L(+/-)-2-amino-3-phosphonopropionic acid (L-AP3). Thus, the current studies suggest that the neuronal EAAs transporter is regulated in different manner by transportable substrates and Glu receptor agonists. The possible involvement of this modulation after certain neurotoxicity insults is discussed.